Microbial growth media provide a nutrient chemical environment for the growth of microorganisms. Microbial growth media are typically composed of water, protein digests and/or extracts, carbohydrates, minerals, a buffer system, and a pH indicator in certain applications. Additional metabolites may be included to influence metabolic pathways. Microbial growth media are made from non-sterile components and are sterilized in the last stages of their fabrication. This is commonly done by heat or filtration.
When the means of sterilization is heat, carbohydrate growth media components are prone to change. It is well known that heating microbial growth media causes carbohydrate degradation and subsequent acidification of growth media. The degree of degradation is a function of the heat applied, the carbohydrate concentration, the starting pH, and the type of carbohydrate present. Carbohydrates can react with amino acids, peptides, and proteins to form brown melanoidins. These are termed Maillard reactions and can result in the destruction of essential nutrients in growth media and color darkening. One class of carbohydrates commonly employed with microbiological growth media is monosaccharides. Monosaccharides, such as the six carbon dextrose, are particularly prone to heat induced Maillard reactions.
U.S. Pat. No. 4,291,122 to Orelski, which is incorporated herein by reference, describes features of microbial growth media as applied to unitary or self-contained biological indicators. Media used in these indicators must support the growth of either Bacillus stearothermophilus in monitoring steam sterilization processes, or Bacillus subtilis in monitoring ethylene oxide sterilization processes. Self-contained biological indicators employ a visual pH indicator that is dissolved in a medium. As the pH of the medium shifts (due to presumed bacterial growth), the color of the media will change, thus simplifying the visual reading of this device.
Growth of Bacillus stearothermophilus causes growth media to become acidic. Bacillus subtilis growth causes growth media to acidify only in the presence of sufficient carbohydrates. Once carbohydrates are consumed, protein components are catabolized. The catabolism of proteins causes growth media to become basic, thus causing the pH indicator that had initially changed color to signify growth, to return to its original color. This process of depleting carbohydrates and consuming excessive protein and causing the pH indicator to return to a false non-growth color is called reversion. One must provide sufficient carbohydrate in media to avoid reversion and at the same time avoid the media degrading Maillard reactions. If a self-contained biological indicator is used to monitor a steam sterilization process, the indicator medium would thus be heated a second time during its actual use in addition to its original sterilization in manufacture. Orelski taught that partitioning carbohydrates from the rest of the liquid medium components avoids the damaging Maillard reactions. In the Orelski teaching, carbohydrates are mixed with the rest of the medium only when the self-contained biological indicator is activated. There is other evidence in the prior art for separate sterilization of carbohydrate components of microbial growth media, but not for this specific application.